Targeting monocytes and macrophages by means of SPECT and PET.

Monocytes have been isolated from patient's blood and directly radiolabelled in vitro using a variety of radiopharmaceuticals such as 99mTc-HMPAO, 111In-oxine, 99mTc-colloids and 18F-FDG. Overall, the best labeling results were obtained using 99mTc-HMPAO. The wide availability of 99mTc and of the ligand HMPAO in kit-formulation makes it the most versatile procedure for imaging localized inflammation using in-vitro labeling. Injection of 99mTc-HMPAO labeled monocytes in adult patients has proven safe with an effective dose of 0.011 mSv/Mbq, equivalent to that of 99mTc-HMPAO labeled mixed white blood cells. Furthermore, in a proof of concept studies, in-vitro labeled monocytes were shown to specifically accumulate in the bowels of patients suffering from inflammatory bowel disease as well as in inflamed joints of rheumatoid arthritis patients. Inversely, the decrease in disease activity of inflamed joints of rheumatoid arthritis patients treated by Adalimumab could not be substantiated using 99mTc-HMPAO labelled monocytes suggesting this type of treatment does not reduce monocyte influx. In spite of their wide availability, in-vitro labeling procedures are cumbersome and time-consuming. Furthermore, cell activation may occur during the labeling process and it cannot be excluded that the radiopharmaceuticals used for labelling interfere with ongoing cellular processes. As such, various authors turned towards the development of radiopharmaceuticals for in-vivo labeling of both monocytes and more importantly macrophages, many of which were subsequently validated in animal models. Targets studied in this regard include amongst others the folate receptor, the mannose receptor, the peripheral benzodiazepine receptor as well as more general characteristics of macrophages such as phagocytosis. Various of these novel molecules appear promising and clinical studies using these radiopharmaceuticals are awaited in the near future. Some of these radiopharmaceuticals also reached the clinical stage, respectively the translocating protein targeting radiopharmaceutical 11C-PK11195 and the folate receptor targeting radiopharmaceutical 99mTc-EC20. Uptake of 11C-PK11195 in inflamed joints and sites of atherosclerosis in patients proved to be directly related to the number of peripheral benzodiazepine binding receptors available as well as to the severity of ongoing inflammation. Comparable results were obtained using 99mTc-EC20 in rheumatoid arthritis patients. In spite of these promising results, additional studies are warranted demonstrating that in vivo, quantitative visualization of monocyte trafficking and accumulation of M1 or M2 macrophage subtypes in sites of ongoing inflammation by means of SPECT and PET will contribute to a better understanding of human inflammatory diseases as well as to diagnosis, treatment planning and the development of targeted treatment strategies.

Detection of macrophages in aortic aneurysms by nanoparticle positron emission tomography-computed tomography.

OBJECTIVE: Current management of aortic aneurysms (AAs) relies primarily on size criteria to determine whether invasive repair is indicated to preempt rupture. We hypothesized that emerging molecular imaging tools could be used to more sensitively gauge local inflammation. Because macrophages are key effector cells that destabilize the extracellular matrix in the arterial wall, it seemed likely that they would represent suitable imaging targets. We here aimed to develop and validate macrophage-targeted nanoparticles labeled with fluorine-18 ((18)F) for positron emission tomography-computed tomography (PET-CT) detection of inflammation in AAs. METHODS AND RESULTS: Aneurysms were induced in apolipoprotein E-/- mice via systemic administration of angiotensin II. Mice were imaged using PET-CT and a monocyte/macrophage-targeted nanoparticle. AAs were detected by contrast-enhanced micro-CT and had a mean diameter of 1.85 ± 0.08 mm, whereas normal aortas measured 1.07 ± 0.03 (P < 0.05). The in vivo PET signal was significantly higher in aneurysms (standard uptake value, 2.46 ± 0.48) compared with wild-type aorta (0.82 ± 0.05, P < 0.05). Validation with scintillation counting, autoradiography, fluorescence, and immunoreactive histology and flow cytometry demonstrated that nanoparticles localized predominantly to monocytes and macrophages within the aneurysmatic wall. CONCLUSIONS: PET-CT imaging with (18)F-labeled nanoparticles allows quantitation of macrophage content in a mouse model of AA.

Labeling monocytes for imaging chronic inflammation.

With growing interest in cell-based scintigraphic diagnosis or therapy monitoring, there is an increasing demand for non-invasive observation and quantification of cell trafficking in the preclinical and clinical setting. Monocytes are members of the human mononuclear phagocyte system originating from a myeloid precursor in the bone. Labeled monocytes are being used for investigation of pathogenesis like atherosclerosis and for monitoring of therapeutic intervention in inflammatory diseases like rheumatoid arthritis. Labeling mononuclear cells at high specific activity without affecting their biological functions allows (delayed) non-invasive imaging with g or PET cameras. Monocytes labeled before their final differentiation into macrophages or dendritic cells may reveal centers of inflammation in a patient and, thereby, contribute to scintigraphic diagnosis. Macrophages or dendritic cells may be in vitro cultured and by means of genetic transformation specified towards specific targets prior to re-injection, an approach with therapeutic potency. This review addresses issues on autologous monocytes, particularly their properties and labeling for non-invasive in vivo radionuclide imaging of chronic inflammation.

Tracking of [18F]FDG-labeled natural killer cells to HER2/neu-positive tumors.

INTRODUCTION: The objective of this study was to label the human natural killer (NK) cell line NK-92 with [(18)F]fluoro-deoxy-glucose (FDG) for subsequent in vivo tracking to HER2/neu-positive tumors. METHODS: NK-92 cells were genetically modified to NK-92-scFv(FRP5)-zeta cells, which express a chimeric antigen receptor that is specific to the tumor-associated ErbB2 (HER2/neu) antigen. NK-92 and NK-92-scFv(FRP5)-zeta cells were labeled with [(18)F]FDG by simple incubation at different settings. Labeling efficiency was evaluated by a gamma counter. Subsequently, [(18)F]FDG-labeled parental NK-92 or NK-92-scFv(FRP5)-zeta cells were intravenously injected into mice with implanted HER2/neu-positive NIH/3T3 tumors. Radioactivity in tumors was quantified by digital autoradiography and correlated with histopathology. RESULTS: The NK-92 and NK-92-scFv(FRP5)-zeta cells could be efficiently labeled with [(18)F]FDG by simple incubation. Optimal labeling efficiencies (80%) were achieved using an incubation period of 60 min and additional insulin (10 IU/ml). After injection of 5x10(6) [(18)F]FDG-labeled NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, digital autoradiography showed an increased uptake of radioactivity in HER2/neu-positive tumors at 60 min postinjection. Conversely, injection of 5x10(6) NK-92 cells not directed against HER2/neu receptors did not result in increased uptake of radioactivity in the tumors. Histopathology confirmed an accumulation of the NK-92-scFv(FRP5)-zeta cells, but not the parental NK cells, in tumor tissues. CONCLUSION: The human NK cell line NK-92 can be directed against HER2/neu antigens by genetic modification. The genetically modified NK cells can be efficiently labeled with [(18)F]FDG, and the accumulation of these labeled NK cells in HER2/neu-positive tumors can be monitored with autoradiography.

Labeling of autologous monocytes with 99mTc-HMPAO at very high specific radioactivity.

Rheumatoid arthritis of joints involves the accumulation of monocyte-derived macrophages in the affected synovial tissue. This process of cell migration can be portrayed scintigraphically in order to monitor noninvasive effects of therapy on the progress of the disease. Scintigraphic detection of inflammation by means of technetium 99m-hexamethylpropylene amine oxime (99mTc-HMPAO)-labeled leukocytes provides a classic example. Present state-of-the-art methods in cell biology allow the isolation of cells like lymphocytes or monocytes, which are less abundant than main blood constituents but, instead, harbor particular functions like specific homing properties. To facilitate scintigraphic imaging of the cell functions involved, the relatively small population of cells must be labeled to radioactive yields as high as possible. We demonstrate that autologous monocytes isolated from 100 ml of peripheral blood can be radiolabeled to a yield of 10 (instead of 1) Bq per cell, allowing scintigraphic analysis of rheumatoid arthritis up to 20 h post injection of patients. The method is based on the instantaneous distribution of lipophilic 99mTc-HMPAO between the hydrophobic inside of cells and the hydrophilic (aqueous) surrounding of cells, followed by decomposition of the radiopharmaceutical into compounds that are unable to cross the cellular membrane. The procedure provides a method of choice for cell-mediated scintigraphy at low availability of cells with the correct homing properties.

[Changes in the activity of the mononuclear phagocytic system in inflammatory diseases and atherosclerosis, as evidenced by radionuclide study].

The purpose of the investigation was to determine the activity of mononuclear phagocytes in inflammatory diseases and atherosclerosis at various predominant sites. The mononuclear phagocytic system was studied using labeled compounds, which determined the functional activity of reticular endothelial elements in all tissues of an organism. The analysis of the results of studying the activity of the mononuclear phagocytic system leads to the conclusion that virtually good agreement of the values of the parameters under study in the groups of patients supports the commonness of the processes of development of inflammatory reactions and atherogenesis.

Relative uptake of technetium 99m stannous colloid by neutrophils and monocytes is altered by gram-negative infection.

Gram-negative infection alters phagocytic cell function; hence, it could affect phagocytic uptake of inorganic colloids by these cells. Neutrophil and monocyte uptake of technetium 99m stannous colloid (99mTc SnC) in whole blood was measured in 10 patients with gram-negative infection (Burkholderia pseudomallei) and 7 controls. Mean uptake per individual neutrophil was reduced in infection. Uptake per monocyte was not significantly different. Blood from six normal individuals was incubated with lysed B. pseudomallei and colloid, which showed reduced neutrophil uptake, but increased monocyte uptake. These results indicate that uptake of 99mTc SnC stannous colloid can be used to measure alteration in phagocytic cell function. They suggest that infection with B. pseudomallei is associated with reduced phagocytosis by individual neutrophils, possibly through toxic effects of bacterial products. This could have immunopathogenic consequences for this gram-negative infection and may explain why it responds to granulocyte colony-stimulating factor.

In vivo tracking for cell therapies.

The success of a particular cellular therapy regime requires the therapeutic agent to migrate expeditiously to the intended target in sufficient numbers and to provoke a desirable response. There are many variables associated with the production, administration and host that need to be investigated to maximize the resulting therapeutic benefit. The large number of factors which may contribute to, or detract from, treatment efficacy can make therapy optimization an arduous procedure. Direct visualization of in vivo migration patterns using nuclear medicine techniques greatly assists the appraisal of the multitude of variables. Conventional radionuclide cell labeling is a proven, simple and sensitive technique which can provide whole body biodistribution information. Labeling with a PET isotope offers greater sensitivity, much improved 3-dimensional resolution and quantification. In general, current efforts are increasingly concentrating on this technology. Imaging studies can supply definitive evidence of successful targeting and allow quantification of the degree of migration to a particular site. Incorporating tracking studies into clinical trials of cell-based therapy at the earliest stage can provide proof of mechanism of the therapy and permit evaluation of the many contributory variables, even on a patient-by-patient basis.

Fluorine-18 radiolabeling of low-density lipoproteins: a potential approach for characterization and differentiation of metabolism of native and oxidized low-density lipoproteins in vivo.

Oxidative modification of low-density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Assessing the metabolic fate of oxidized LDL (oxLDL) in vivo with radiotracer techniques is hindered by the lack of suitable sensitive and specific radiolabeling methods. We evaluated an improved methodology based on the radiolabeling of native LDL (nLDL) and oxLDL with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). We investigated whether radiolabeling of LDL induces adverse structural modifications. Results suggest that radiolabeling of both nLDL and oxLDL using [(18)F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively. Thus, radiolabeling of LDL using [(18)F]SFB could prove to be a promising approach for studying the kinetics of oxLDL in vivo.

Characterization of 18F-FDG uptake in human endothelial cells in vitro.

UNLABELLED: The contribution of (18)F-FDG uptake by endothelial cells to uptake values measured by PET in various tissues is as yet unclear. We therefore sought to characterize (18)F-FDG uptake in an in vitro model of human endothelial cells. METHODS: Commercially obtained human umbilical vein endothelial cells (HUVECs) were seeded in 6-multiwell plates 48-96 h before incubation with 1-2 MBq (18)F-FDG per well. Radioactivity measurements were performed after washing and mechanical dissolvation of the cellular monolayers. Cellular (18)F-FDG uptake was referred to protein concentration. This experimental protocol was subsequently varied to study the effect of different parameters of interest. Furthermore, radio-thin-layer chromatography was used to identify intracellular (18)F-FDG metabolites. (18)F-FDG uptake in HUVECs was compared with that by a human monocyte-macrophage (HMM) preparation and by glioblastoma cells (GLIOs) under identical experimental conditions. RESULTS: (18)F-FDG accumulated in HUVECs in a time-dependent manner and was trapped mainly as (18)F-FDG-6-phosphate and (18)F-FDG-1,6-diphosphate. Unlabeled glucose and cytochalasin B competitively inhibited (18)F-FDG uptake, whereas phlorizin had no significant effect. Glucose deprivation significantly enhanced (18)F-FDG uptake by a factor of 2.7, whereas sodium depletion had no significant influence. HUVECs treated with vascular endothelial growth factor (VEGF) showed a significant 82% increase in (18)F-FDG accumulation after a 2-h exposure to 50 ng/mL VEGF. (18)F-FDG uptake in HUVECs was significantly higher than that in HMMs and in the range of the uptake values measured in GLIOs. CONCLUSION: (18)F-FDG accumulates in HUVECs by mechanisms analogous to those in neoplastic cells or neurons. VEGF significantly stimulates endothelial (18)F-FDG uptake. The observed differences in (18)F-FDG uptake between HUVECs, HMMs, and GLIOs are difficult to extrapolate to in vivo conditions but stimulate further studies on the contribution of endothelial (18)F-FDG uptake to the overall uptake of that tracer in neoplastic or vascular lesions.

Augmented 18F-FDG uptake in activated monocytes occurs during the priming process and involves tyrosine kinases and protein kinase C.

UNLABELLED: Activated monocytes with a high (18)F-FDG accumulation can affect the results of clinical PET studies. To better understand the mechanisms regulating monocytic (18)F-FDG uptake, we investigated the effect of priming and respiratory-burst generation and further evaluated the role of potential protein kinase pathways. METHODS: Purified human monocytes were primed with interferon-gamma (IFN-gamma), and respiratory burst was generated by stimulation of primed cells with phorbol-12-myristate-13-acetate (PMA). Oxygen-intermediate generation was assessed by luminescence measurements after the addition of lucigenin. (18)F-FDG uptake after 30 min of incubation was measured for unprimed control cells, primed cells, and PMA-stimulated cells. The role of protein kinases was investigated using respective inhibitors. RESULTS: PMA stimulation of primed monocytes dramatically increased oxygen-intermediate generation, leading to a 42.2 +/- 1.1 fold higher level of cumulative luminescence compared with unprimed control cells, whereas IFN-gamma priming alone resulted in low luminescence levels (13.9% +/- 4.6% of PMA-stimulated cells). In contrast, priming alone was sufficient to augment monocytic (18)F-FDG uptake to 273.3% +/- 16.7% of control levels (P < 0.001), and it was not further increased by PMA stimulation. The tyrosine kinase inhibitor, genistein, and the specific protein kinase C inhibitor, staurosporine, completely abolished the priming-induced enhancement of (18)F-FDG uptake and lowered uptake to control levels. Under the same conditions, wortmannin, a phosphatidylinositol 3 kinase (PI3 kinase)-specific inhibitor, and cycloheximide, a protein synthesis inhibitor, were associated with only minor reductions in the enhanced-uptake effect of priming. CONCLUSION: IFN-gamma priming alone, without stimulation of respiratory-burst activity, is sufficient to induce maximal augmentation of (18)F-FDG uptake in monocytes. Furthermore, this metabolic effect appears to involve tyrosine kinases and the protein kinase C pathway but is independent of the PI3 kinase pathway.

Uptake of [18F]fluorodeoxyglucose in human monocyte-macrophages in vitro.

The fact that fluorine-18 fluorodeoxyglucose ([(18)F]FDG) accumulates in inflammatory lesions as well as in tumours reduces the diagnostic specificity of positron emission tomography (PET) in oncology. The aim of this study was to characterise the uptake of [(18)F]FDG in isolated human monocyte-macrophages (HMMs) in vitro in comparison with that in human glioblastoma (GLI) and pancreatic carcinoma cells (PAN). The purity of HMM preparations was determined by immunohistochemical staining and their functional integrity was assessed by long-term incubation with iodine-131 acetylated bovine serum albumin. [(18)F]FDG uptake in HMMs was quantified as percent of whole [(18)F]FDG activity per well (% ID) or as % ID in relation to total protein mass. [(18)F]FDG uptake in HMMs significantly increased with culture duration, yielding 7.5%+/-0.9% (% ID/100 micro g) at day 14. Stimulation by lipopolysaccharide further enhanced [(18)F]FDG uptake in HMMs by a factor of 2. [(18)F]FDG uptake significantly decreased with increasing glucose concentration in the medium. Radio-thin layer chromatography of intracellular metabolites revealed that [(18)F]FDG was trapped by HMMs mainly as [(18)F]FDG-6-phosphate and [(18)F]FDG-1,6-diphosphate. [(18)F]FDG uptake was in the range of uptake values measured in GLI and PAN. By accumulating [(18)F]FDG in a manner analogous to uptake by tumour cells, activated HMMs may contribute to the [(18)F]FDG uptake values measured by PET in neoplasms.

Use of insulin to improve [18 F]fluorodeoxyglucose labelling and retention for in vivo positron emission tomography imaging of monocyte trafficking.

While 18F-FDG labelling of monocytes would allow in vivo trafficking with positron emission tomography (PET), present methods suffer from poor retention of radioactivity. We investigated the feasibility of utilizing insulin for improved [18F]fluorodeoxyglucose (18F-FDG) labelling. Separated human monocytes and lymphocytes were labelled with 18F-FDG with or without 3 h insulin pre-incubation. Insulin had no effect on lymphocyte labelling (21.4+/-0.8% vs 20.8+/-1.1% efficiency, P=NS). However, for monocytes, insulin pre-incubation led to a 169+/-9% increase in labelling efficiency (19.3+/-4.1 vs 32.5+/-1.8, P<0.05), without significant effects on cell activation or viability. Moreover, while only 57.7+/-4.8% and 40.4+/-5.6% of the 18F-FDG was retained at 1 and 3 h for controls, the retention rate increased to 91.6+/-2.1% (P=0.01) and 86.5+/-1.9% (P<0.01) after insulin pre-incubation. Improved 18F-FDG retention was accompanied by a 70.3+/-7.4% decrease in glucose-6-phosphatase activity (P=0.02). PET imaging of rats showing hepatic ischaemia-reperfusion injury demonstrated higher liver uptake for monocytes labelled after insulin treatment. Thus, insulin improves monocytic 18F-FDG uptake and retention, and may provide a feasible labelling method for PET imaging.

Imaging macrophages and the apoptosis of granulocytes in a rodent model of subacute and chronic abscesses with radiolabeled monocyte chemotactic peptide-1 and annexin V.

Monocytes/macrophages (Mphis), the predominant cell types in subacute and chronic inflammation, are attracted to and activated by monocyte chemotactic peptide-1 (MCP-1). Mphis promote the resolution of inflammation through the induction of apoptosis and phagocytosis of senescent (spent) and bystander (superfluous) granulocytes. We wished to determine whether MCP-1, which selectively binds to Mphis, could be used to image subacute and chronic inflammation. We also sought to image granulocyte apoptosis within these lesions with technetium-99m labeled annexin V, a marker of apoptotic cells. Sterile inflammation was induced in 45 12-week-old male Sprague-Dawley rats by deep intramuscular injection of turpentine into the right thigh. Groups of four to six animals were then imaged 1 h after tail vein injection of 37-148 MBq (1-4 mCi) of 99mTc-labeled MCP-1 or annexin V 1-14 days after turpentine treatment. Image analysis showed significantly greater activity of both MCP-1 and annexin V in inflamed thighs than in control thighs (165%-290% and 188%-313%, respectively; P<0.01) on days 1-5 after turpentine injection. Dual autoradiography in animals co-injected with iodine-125 labeled bovine serum albumin on days 1 and 4 showed specific location of MCP-1 to infiltrating Mphis while annexin V localized to focal zones of apoptosis within granulocytic infiltrates adjacent to abscess cavities. Scintillation well counting on day 5 demonstrated significantly higher (P<0.005) ratios of abscess to control thigh specific activities for MCP-1 (5.83+/-2.17) and annexin V (9.24 +/- 2.8) as compared to 125I-labeled bovine serum albumin (3.11 +/- 0.65). No significant increases in uptake were noted at imaging or ex vivo analyses on days 13 and 14, when lesions were predominately fibrotic. It is concluded that 99mTc-labeled MCP-1 and 99mTc-labeled annexin V both localize in zones of subacute inflammation, reflecting the density of Mphis and the incidence of apoptotic granulocytes, respectively. These agents may be useful in the characterization of subacute inflammation.

Do high glucose levels have differential effect on FDG uptake in inflammatory and malignant disorders?

BACKGROUND: The association of hyperglycaemia with reduced fluorodeoxyglucose (FDG) uptake by tumour cells is well established. Therefore, it is standard practice that all patients must fast for at least several hours prior to FDG positron emission tomography (PET) imaging. However, the effect of hyperglycaemia on FDG uptake by inflammatory and infectious lesions is unknown. The aim of this study was to investigate this important issue. METHODS: For in vitro studies human mononuclear cells were isolated from 12 normal volunteers and FDG uptake was determined in medium containing differing concentrations of glucose. FDG uptake by human mesothelioma cells was also measured for comparison. For studies involving patients, 416 FDG PET scans of patients with confirmed malignancy (n=321) or benign lesions (n=95) were reviewed retrospectively. The relationship between serum glucose level and FDG uptake by the lesions was assessed utilizing the standardized uptake value (SUV) technique. RESULTS: In the in vitro studies, while FDG uptake by mesothelioma cells decreased as glucose concentration increased, there was no differential uptake of FDG uptake by mononuclear cells at glucose concentrations less than 250 mg x dl(-1). In clinical patients, FDG uptake by malignant lesions was slightly, but negatively affected by serum glucose level (r= -0.21, P<0.01) (glucose range 49-187 mg x dl(-1)). In contrast, FDG uptake by inflammatory lesions was positively associated with serum glucose level (r=0.43, P<0.01) (glucose range 54-215 mg x dl(-1)). DISCUSSION AND CONCLUSION: While the degree of FDG uptake is primarily influenced by the nature of the underlying lesion, serum glucose concentration appears to have a small effect on FDG uptake, which differs between malignant disorders and inflammatory processes. Our data suggest that below a certain level, elevated glucose concentration might not have a negative effect on FDG uptake in inflammatory cells, contrary to that observed in malignant disorders.

Effect of ultrasound on the production of IL-8, basic FGF and VEGF.

Therapeutic angiogenesis is the controlled induction or stimulation of new blood vessel formation to reduce unfavourable tissue effects caused by local hypoxia and to enhance tissue repair. The effects of ultrasound on wound healing, chronic ulcers, fracture healing and osteoradionecrosis may be explained by the enhancement of angiogenesis. The aim of this study was to identify which cytokines and angiogenesis factors are induced by ultrasound in vitro. Two ultrasound machines were evaluated, a "traditional" (1 MHz, pulsed 1:4, tested at four intensities), and a "long wave" machine (45 kHz, continuous, also tested at four intensities). The ultrasound was applied to human mandibular osteoblasts, gingival fibroblasts and peripheral blood mononuclear cells (monocytes). The following cytokines and angiogenesis factors were assayed by ELISA techniques: interleukin-1beta(IL-1beta), IL-6, tumour necrosis factor alpha (TNF-alpha), IL-8, fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).A slight stimulation of IL-1beta was noted in all cell types. There was no difference in the IL-6 and TNF-alpha levels. The angiogenesis-related cytokines, IL-8 and bFGF, were significantly stimulated in osteoblasts, and VEGF was significantly stimulated in all cell types. Both ultrasound machines produced similar results, and the optimum intensities were 0.1 and 0. 4 W/cm2 (SATA) with 1 MHz ultrasound, and 15 and 30 mW/cm2 (SATA) with 45 kHz ultrasound.The results show that therapeutic ultrasound stimulates the production of angiogenic factors such as IL-8, bFGF and VEGF. This may be one of the mechanisms through which therapeutic ultrasound induces angiogenesis and healing.

A new approach to labeling cells with technetium-99m, Part I. Preparation of modified polylysine and in vitro cell labeling.

A method for labeling cells with technetium-99m via hydrophilic, polycationic poly D-lysine modified by N-acetyl homocysteine has been developed. The modified polylysine (MPL) is labeled with 99mTc in > 95% yield and is stable for > 12 h. Maximum cell labeling is achieved by a 1-h incubation at room temperature with isolated leukocytes, granulocytes and peripheral blood mononuclear cells attaining 60-75% 99mTc incorporation, and red blood cells 35%. Ninety-two percent of the label is retained by leukocytes after a 1-h incubation at room temperature in 50% serum. The cell uptake of 99mTc-MPL is affected by the presence of negatively charged species in the medium; the inhibitory effects of 5% serum or serum albumin can be reversed by increasing the concentration of 99mTc-MPL, while those of heparin are not.

Direct evidence of monocyte recruitment to inflammatory bowel disease mucosa.

Alterations in phenotype and function of intestinal macrophages occur in inflammatory bowel disease (IBD) but it is unclear whether these changes result from the recruitment of circulating monocytes to the intestine or from proliferation of resident intestinal macrophages. We sought to demonstrate the arrival of blood monocytes, the precursors of macrophages, in IBD mucosa. Peripheral blood mononuclear cells were isolated from 23 patients with clinically active intestinal inflammation (13 Crohn's disease, eight ulcerative colitis, two infective colitis), then radiolabelled with 99mtechnetium (Tc)-stannous colloid (n = 13) or 111indium (In)-oxine (n = 10) before re-injection and abdominal scanning. Four patients had demonstrable intestinal monocyte uptake using [99mTc]-stannous colloid, while six [111In]-oxine-labelled monocyte scans were positive. Uptake sites correlated with actively inflamed regions. Patients demonstrating monocyte uptake had been treated with corticosteroids for a significantly (P < 0.02) shorter duration (median 3 vs 20 days) than those with negative scans. There was no significant difference between positive and negative scans for disease category, clinical or histological disease, activity, or radioisotope used. Biopsies of inflamed mucosa from two patients suffering ulcerative colitis who had positive scans showed a high proportion of CD14-positive macrophages, 4-9% of which contained autoradiographic grains. These results demonstrate that blood monocytes are recruited to the mucosa of actively inflamed bowel, and suggest that this process may be inhibited by corticosteroids. Moreover, the phenotype of the recently-arrived monocytes indicates their susceptibility to stimulation by lipopolysaccharide, and suggests a mechanism for the continuing inflammation in the bacterial product-rich milieu of IBD.